Iskra Katic et al. (2003) International Worm Meeting "Characterization of sel-6, a suppressor of lin-12gain-of-function mutants"

Iskra Katic, Iva Greenwald

[International Worm Meeting, 2003]

LIN-12/Notch proteins mediate multiple cell-cell interactions that specify cell fate. Isolation of extragenic suppressors and enhancers of various lin-12alleles has been a powerful method for identifying factors that influence LIN-12 mediated signaling.In a screeen for suppressors of lin-12gain of function mutants, two sel-6alleles were identified (F.E. Tax, J.H. Thomas, E.L. Ferguson and H.R. Horvitz, 1997). We positionally cloned sel-6and found it to encode a protein that contains a potentially informative sequence motif and is conserved in higher eukaryotes. We are performing genetic and molecular analyses to address the role of SEL-6 in the LIN-12/GLP-1 signaling pathways, in the AC/VU as well as other cell fate decisions during C. elegansdevelopment. We will report on our progress at the meeting.

Iskra Katic et al. (2004) East Coast Worm Meeting "Characterization of sel-6, a suppressor of lin-12 gain-of-function mutants"

Iskra Katic, Iva Greenwald

[East Coast Worm Meeting, 2004]

LIN-12/Notch proteins are receptors that mediate cell-cell interactions that specify cell fate. Isolation of extragenic suppressors and enhancers of various lin-12 alleles has been a powerful method for identifying core components of the signal transduction machinery and factors that influence LIN-12 activity. Suppressors of a lin-12 gain-of-function mutation previously defined the sel-6 gene (Tax et al., 1997). We positionally cloned sel-6 and found it to encode a protein containing a AAA domain (ATPases associated with diverse cellular activities) and is conserved in higher eukaryotes. We have found that sel-6 is necessary for proper embryonic and larval development, and that the suppressor alleles appear to be hypomorphs. We are performing genetic and molecular analyses to address the role of SEL-6 in the LIN-12/GLP-1 signaling pathways, in the AC/VU as well as other cell fate decisions during C. elegans development. We will report on our progress at the meeting.

Iskra Katic et al. (2005) International Worm Meeting "New positive regulators of lin-12 activity in Caenorhabditis elegans include genes that encode glycosphingolipid biosynthesis enzymes"

Iskra Katic, Laura Vallier, Iva Greenwald

[International Worm Meeting, 2005]

Screens for suppressors of lin-12 hypermorphic alleles in C. elegans have identified core components and modulators of the LIN-12/Notch signaling pathway. We recovered alleles of six new genes from a screen for suppressors of the egg-laying defect associated with a lin-12 hypermorph. The molecular identification of one of the new suppressor genes revealed it as bre-5, the C. elegans homolog of D. melanogaster brainiac, which had also been identified in screens for mutations that confer resistance to Bt toxin in C. elegans. BRE-5/Brainiac catalyzes a step in the synthesis of glycosphingolipids, components of lipid rafts that are thought to act as platforms for association among certain kinds of membrane-bound proteins. Reducing the activity of several other genes involved in glycosphingolipid biosynthesis also suppresses the effects of constitutive lin-12 activity. Our results suggest that a role for glycosphingolipids as positive regulators of lin-12/Notch activity is conserved.

Venz, Richard et al. (2021) International Worm Meeting "End-of-life targeted auxin-mediated degradation of DAF-2 Insulin/IGF-1 receptor promotes longevity free from growth-related pathologies"

Venz, Richard, Ewald, Collin, Pekec, Tina, Katic, Iskra, Ciosk, Rafal

[International Worm Meeting, 2021]

Preferably, lifespan-extending therapies should work when applied late in life without causing undesired pathologies. However, identifying lifespan-extending interventions that are effective late in life and which avoid undesired secondary pathologies remains elusive. Reducing Insulin/IGF-1 signaling (IIS) increases lifespan across species, but the effects of reduced IIS interventions in extreme geriatric ages remains unknown. Using the nematode C. elegans, we engineered the conditional depletion of the DAF-2/insulin/IGF-1 transmembrane receptor using an auxin-inducible degradation (AID) system that allows for the temporal and spatial reduction in DAF-2 protein levels at time points after which interventions such as RNAi may lose efficacy. Using this system, we found that AID-mediated depletion of DAF-2 protein efficiently extends animal lifespan. Depletion of DAF-2 during early adulthood resulted in multiple adverse phenotypes, including growth retardation, germline shrinkage, egg-retention, and reducing offspring. By contrast, however, AID-mediated depletion of DAF-2 specifically in the intestine resulted in an extension of lifespan without these deleterious effects. Importantly, AID-mediated depletion of DAF-2 protein in animals past their median lifespan allowed for an extension of lifespan without affecting growth or behavioral capacity. Thus, both late-in-life targeting and tissue-specific targeting of IIS minimize the deleterious effects typically seen with interventions that reduced IIS, suggesting potential therapeutic methods by which longevity and healthspan can be increased in even geriatric populations.

Paula Checchi et al. (2006) Development & Evolution Meeting "emb-4 is a Highly Conserved Gene with a Role in Germline-Specific Chromatin Remodeling during C. elegans Embryogenesis"

William Kelly, Paula Checchi

[Development & Evolution Meeting, 2006]

Early embryogenesis in C. elegans is characterized by a series of asymmetric cellular divisions, resulting in somatic lineages sequentially separating from "P"-germ blastomeres. A number of protective mechanisms exist that appear to shield germline blastomeres from inductive signals that specify the fates of somatic cells. In one mechanism, germ cells are kept transcriptionally quiescent by the maternally loaded CCCH protein PIE-1. However, upon the birth of the germ cell precursors Z2 and Z3, PIE-1 disappears, yet Z2/Z3 remain quiescent. The mechanisms controlling the temporal regulation of PIE-1 in the nascent germ cells as well as subsequent repressive processes are poorly understood. We have previously demonstrated that there is a chromatin-based repression that succeeds PIE-1 degradation (Schaner et al., 2003). The chromatin in the primordial germ cells Z2/Z3 loses certain histone modifications including histone H3 K4 dimethylation, a conserved marker for transcriptionally competent chromatin. Here we show that mutations in emb-4 cause defects in both PIE-1 degradation and in germline-specific chromatin remodeling. We show that, as in early somatic blastomeres (Seydoux lab), the degradation of PIE-1 in Z2/Z3 is dependent upon the CCCH binding protein ZIF-1. In the absence of either zif-1 or emb-4, embryos abnormally retain PIE-1 in Z2/Z3. emb-4 is allelic to two previously identified mutants including sel-6, a suppressor of the lin-12(d) (Notch) mutant (Iskra Katic and Iva Greenwald, personal communication), and we have demonstrated that emb-4 encodes a highly conserved, novel protein with orthologs in fly, mouse, and human, for which functions have not been previously described. Currently we are investigating the molecular and genetic mechanisms that control PIE-1 degradation and germline-specific chromatin remodeling during embryogenesis.

Paula M Checchi et al. (2005) International Worm Meeting "emb-4 Encodes a Highly Conserved Gene with a Role in Germline-Specific Chromatin Remodeling and Cell Fate Specification During C. elegans Embryogenesis."

Paula M Checchi, William G Kelly, Christine E Schaner

[International Worm Meeting, 2005]

In C. elegans, early embryogenesis is partially orchestrated by a well-characterized series of asymmetric cellular divisions, resulting in somatic lineages sequentially separating from P-germ blastomeres. In the early embryo, a number of protective mechanisms exist that appear to shield germline blastomeres from inductive signals that specify the fates of somatic cells. Specifically, germ cells are kept transcriptionally quiescent by the activity of the maternally loaded CCCH protein PIE-1. However, upon the birth of the germ cell precursors Z2 and Z3, PIE-1 disappears. The mechanisms controlling the temporal regulation of PIE-1 in the nascent germ cells, as well as post-PIE-1 repressive processes, are poorly understood. We have previously demonstrated that there is a chromatin based repressive mechanism that succeeds PIE-1 degradation, effectively marking and maintaining the germ lineage. The primordial germ cells Z2/Z3 lose the histone H3 modification K4 dimethylation, a conserved marker for transcriptionally competent chromatin. We have demonstrated that germline-specific chromatin remodeling is defective in the temperature sensitive embryonic arrest mutant emb-4. In addition, late-stage emb-4 embryos fail to differentiate properly and arrest as masses of poorly differentiated cells. This defect is accompanied by ectopic expression of the germline-specific marker PGL-1, suggesting an involvement of emb-4 in the repression of germ cell fate in the soma. In collaboration with the Greenwald lab, we have shown that emb-4 is allelic to two previously identified mutants including sel-6, a suppressor of the lin-12(d) (Notch) mutant (Iskra Katic and Iva Greenwald, personal communication). The emb-4/sel-6 gene encodes a novel, highly conserved protein with orthologs in fly, mouse and human. Currently we are investigating the molecular and genetic mechanisms by which EMB-4/SEL-6 dictates germline-specific chromatin remodeling and cell fate specification in the embryo.

Checchi PM et al. (2006) Genetics "emb-4 is a conserved gene required for efficient germline-specific chromatin remodeling during Caenorhabditis elegans embryogenesis."

Checchi PM, Kelly WG

[Genetics, 2006]

IIn C. elegans, germline blastomeres are initially kept transcriptionally quiescent by the maternally loaded CCCH zinc-finger protein PIE-1. PIE-1 disappears upon the birth of the primordial germ cells Z2 and Z3, yet these cells appear to remain quiescent. We have previously demonstrated that there is a chromatin-based repression that succeeds PIE-1 degradation. The chromatin in Z2/Z3 loses certain histone modifications including histone H3 lysine 4 dimethylation (H3K4me2), a conserved marker for transcriptionally competent chromatin. We find that mutations in the maternal effect gene emb-4 cause defects in both PIE-1 degradation and in germline-specific chromatin remodeling. emb-4 encodes a highly conserved protein with orthologs in fly, mouse, and human and has a subtle role in Notch signaling (KATIC and GREENWALD, accompanying manuscript). The embryonic phenotype of emb-4 is consistent with a defect in the efficient and timely activation of developmental programs, including germline chromatin remodeling. We also find that, as in early somatic blastomeres, the degradation of PIE-1 in Z2/Z3 is facilitated by zinc finger-interacting protein ZIF-1, and in the absence of either zif-1 or emb-4 PIE-1 is abnormally retained in Z2/Z3.

Jiabin Chen et al. (2001) International C. elegans Meeting "Characterization of a lin-12(d) suppressor, sel-7"

Jiabin Chen, Xiajun Li, Iva Greenwald

[International C. elegans Meeting, 2001]

LIN-12/Notch proteins function as transmembrane receptors for intercellular signals during development. They are activated by binding of their ligands, DSL (Delta/Serrate/LAG-2) proteins. Ligand binding triggers at least one proteolytic processing event in the extracellular domain and then SEL-12/presenilin-dependent proteolysis within the transmembrane domain. This latter event releases the intracellular domain, which translocates to the nucleus and activates transcription of target genes in a complex with LAG-1, SEL-8, and presumably other proteins. The anchor cell (AC)/ventral uterine precursor cell (VU) fate decision has served as an important model for LIN-12/Notch cell signalling events. Constitutive activation of LIN-12, by lin-12(d) mutations, causes the presumptive AC to be transformed into a VU. As no AC is made, no vulva is induced, leading to an egg-laying defective (Egl) phenotype. Reversion of the Egl phenotype yields intragenic and extragenic suppressor mutations. Such reversion screens have been carried out on a large scale (Tax et al., 1997; L. Vallier, I. Katic, J. Chen, and I. Greenwald, unpublished observations). Many of the extragenic suppressors, sel genes (suppressor/enhancer of lin-12), have proven to be critical components of LIN-12/Notch signal transduction. An extragenic suppressor mutation, n1253, isolated by Tax et al. 1997 defined the sel-7 gene; our lab has identified an additional allele, sel-7(ar516). We have characterized the genetic interaction of these alleles with various alleles of lin-12 and glp-1. We have also cloned sel-7, and found that it encodes a novel protein. We are currently exploring its expression and subcellular localization pattern, and hope to have some insight as to its function in LIN-12/Notch signalling by the meeting.

Olmedo, Maria et al. (2015) International Worm Meeting "Dissection of the temperature control of C. elegans larval development using a novel high-throughput method."

Geibel, Mirjam, Merrow, Martha, Olmedo, Maria, Artal-Sanz, Marta

[International Worm Meeting, 2015]

C. elegans postembryonic development is characterised by four discrete larval stages. The transitions between larval stages entail a series of events including the formation of a new cuticle and escape from the old one, cessation of pharyngeal pumping and behavioral quiescence. The entire process is called molting and robust waves of gene expression cycle with these processes. These events are used as hallmarks to study the timing of the developmental process, typically, by visual inspection. This method is time consuming and unsuitable for screening large numbers of animals.We have applied a method based on bioluminescence to measure the alternation of larval stages and molts, extracting information about the duration of each stage of development. We have validated this method with a lin-42 mutant that shows slower development than wild-type animals (1) and with a daf-2 mutant that shows an extended second larval stage (2).Development in C. elegans is controlled by genetic and environmental factors. Temperature regulates the speed of embryonic and post-embryonic development in many ectothermic organisms. The effect of temperature on embryonic development in C. elegans and Drosophila (3, 4), and on postembryonic development in Drosophila (3) have been described. However, a detailed analysis of larval developmental at different temperatures has never been shown for C. elegans. We have determined the duration of each molt and each larval stage at temperatures between 10.3 C and 27.5 C. The speed of development increases with temperature in a non-linear manner. At low temperatures, the duration of development follows the Arrhenius equation. At higher temperatures, the speed of development deviates from Arrhenius. Furthermore, we have show that this difference stems mainly from the molts. We are currently investigating the implication of thermosensory pathways in the control of developmental speed.1. Monsalve, Van Buskirk and Frand, Current Biology 21, 2033-2045 (2011).2. Ruaud, Katic and Bessereau, Genetics 187, 337-343 (2011).3. Powsner, Physiological Zoology 8, 474-520 (1935).4. Begasse, et al., Cell Reports 10, 647-653 (2015).

Jiabin Chen et al. (2003) International Worm Meeting "sel-7 and sel-13, Suppressors of lin-12(d), Encode Novel Nuclear Proteins"

Hanna FARES, Iva Greenwald, Jiabin Chen, Xiajun Li

[International Worm Meeting, 2003]

LIN-12/Notch proteins function as transmembrane receptors for intercellular signals during development. They are activated by binding of their ligands, DSL (Delta/Serrate/LAG-2) proteins. Ligand binding triggers at least one proteolytic processing event in the extracellular domain and then SEL-12/presenilin-dependent proteolysis within the transmembrane domain. This latter event releases the intracellular domain, which translocates to the nucleus and activates transcription of target genes in a complex with LAG-1, SEL-8, and presumably other proteins. The anchor cell (AC)/ventral uterine precursor cell (VU) fate decision has served as an important model for LIN-12/Notch cell signalling events. Constitutive activation of LIN-12, by lin-12(d) mutations, causes the presumptive AC to be transformed into a VU, resulting in a 0AC-Egl (egg-laying defective) phenotype. Reversion of the 0AC phenotype yields intragenic and extragenic suppressor mutations. Such reversion screens have been carried out on a large scale, using EMS, and more recently, RNAi (Greenwald et al., 1983, Tax et al., 1997; L. Vallier, I. Katic, J. C., C. Bais and I. G., unpublished observations). Extragenic suppressors have defined sel genes (suppressor/enhancer of lin-12 ), and many sel genes have proven to encode critical components of LIN-12/Notch signal transduction. We have been focusing on two genes, sel-7 and sel-13. Loss of sel-7 activity does not cause any overt phenotypes in an otherwise wild-type background. However, sel-7 reduces lin-12 activity in the AC/VU decision, and also affects a subset of other cell fate decisions mediated by lin-12 and glp-1. sel-7encodes a novel protein that has clear orthologs in other nematodes but not in other phyla. A GFP-tagged SEL-7 protein localizes to the nucleus. SEL-7 itself does not appear to interact physically with known nuclear components of the LIN-12 pathway, but another novel protein that interacts with SEL-7 also appears to interact with SEL-8 (a known nuclear component). This result, along with the genetic interactions we have observed, would be consistent with a role for SEL-7 as part of the transcriptional activation complex formed upon LIN-12 activation. Our genetic and molecular analysis of sel-13 has paralleled our analysis of sel-7. Loss of sel-13 activity also reduces lin-12 activity in the AC/VU decision. SEL-13 also appears to be a nuclear protein, but we have not yet been able to detect any physical interactions with known nuclear components of the LIN-12 pathway.